Alpha-1,3 fucosyltransferases and expression systems for making and expressing them

ABSTRACT

A bacterial α1,3-fucosyltransferase gene and deduced amino acid sequence is provided. The gene is useful for preparing α1,3-fucosyltransferase polypeptide, and active fragment thereof, which can be used in the production of oligosaccharides such as Lewis X, Lewis Y, and siayl Lewis X, which are structurally similar to certain tumor-associated carbohydrate antigens found in mammals. These product glycoconjugates also have research and diagnostic utility in the development of assays to detect mammalian tumors. In addition the polypeptide of the invention can be used to develop diagnostic and research assays to determine the presence of  H. pylori  in human specimens.

[0001] This application claims priority to U.S. Patent ApplicationSerial No. 60/048,857, filed Jun. 6, 1997, which is incorporated hereinby reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field ofα1,3-fucosyltransferases and, more specifically, toα1,3-fucosyltransferase polypeptides which are transmembranesegment-free.

BACKGROUND OF THE INVENTION

[0003]Helicobacter pylori is an important human pathogen which causesboth gastric and duodenal ulcers and has also been associated withgastric cancer and lymphoma. This microorganism has been shown toexpress cell surface glycoconjugates including Lewis X, Lewis Y, andsialyl Lewis X. These bacterial oligosaccharides are structurallysimilar to tumor-associated carbohydrate antigens found in mammals.

[0004] The presence of H. pylori isolate has been associated with anincreased risk for development of gastric cancer (Wirth, H. -P., Yang,M., Karita, M., and Blaser, M. J. (1996) Infect. Immun. 64, 4598-4605).This pathogen is highly adapted to colonize human gastric mucosa and mayremain in the stomach with or without causing symptoms for many years.Although H. pylori elicits local as well as systemic antibody responses,it escapes elimination by the host immune response due to itssequestered habitation within human gastric mucosa. Another mechanism bywhich H. pylori may protect itself from the action of the host immuneresponse is the production of surface antigens mimicking those in thehost.

[0005] In mammalian cells the enzyme α(1,3/1,4)-fucosyltransferase(namely FucT) catalyzes the last step in the synthesis of twocarbohydrate structures, Galβ1-4[Fucα1-3]GlcNAc (Lewis X, Le^(x) forshort) or NeuAcα2-3-Galβ1-4[Fucα1-3]GlcNAc (sialyl Lewis X, sLe^(x) forshort). (Lowe et al., 1990, Cell 57: 475-484.; Kukowska-Latallo et al.,1990, Genes & Development 4:1288-1303.) Cell surface α(1,3)- andα(1,2)-fucosylated oligosaccharides, that is, Lewis X (Le^(x)), sialylLewis X (sLe^(x)) and Lewis Y (Le^(y)), are present on both eukaryoticand microbial cell surfaces. In mammals, Le^(x) is a stage-specificembryonic antigen, however, Le^(x), sLe^(x) and Le^(y) are also regardedas tumor-associated markers. The biological functions of these bacterialoligosaccharide structures are not fully understood. It has beensuggested that such glycoconjugates produced by H. pylori, may mimichost cell antigens and could mask the bacterium from the host immuneresponse. It is also possible that these bacterial Lewis antigens coulddown regulate the host T-cell response. Therefore, production of suchantigens may contribute to colonization and long-term infection of thestomach by H. pylori.

[0006] Presently, use of carbohydrates as potential therapeutic drugshas become popular in the field of medical chemistry. In addition,qualitative and quantitative carbohydrates including Le^(x), Le^(y) andsLe^(x) are also required as reagents for assaying the enzymes which areinvolved in the biosynthesis of glycoconjugates in cells. Le^(x), Le^(y)and sLe^(x) products which are commercially available are chemicallysynthesized. However, synthesis of these products gives rise to severallimitations such as time-consuming, complicated procedures and lowyields. Although several mammalian fucosyltransferases have been clonedand expressed, enzymatic synthesis of Le^(x), Le^(y) and sLe^(x)products for a commercial purpose has not been reported.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the discovery of a novelα1,3-fucosyltransferase polypeptide and gene which encodes thepolypeptide. The present invention includes a novel nucleic acidsequence of α1,3-fucosyltransferase polypeptide which is useful in thedetection and synthesis of α1,3-fucosyltransferase polypeptide. Inanother embodiment, the invention provides a method of using the novelα1,3-fucosyltransferase to synthesize oligosaccharides such as Le^(x),Le^(y) and sLe^(x).

[0008] In another embodiment the invention provides the novelpolypeptide of α1,3-fucosyltransferase which is useful in thedevelopment of antibodies to α1,3-fucosyltransferase.

[0009] In another embodiment, the novel polypeptide ofα1,3-fucosyltransferase has a carboxyl terminal ˜100 amino acids inlength having therein a heptad repeat of X₁X₂LRX₃X₄Y, wherein X₁ is D orN; X₂ is D or N; X₃ is I, V or A; X₄is N or D in another embodiment, theα1,3-fucosyltransferase is a peptide selected from SEQ ID NO: 1, SEQ IDNO: 2, and SEQ ID NO: 3. In another embodiment theα1,3-fucosyltransferase may have a variable number of heptad repeats.

[0010] Further provided is a method for producingα1,3-fucosyltransferase. The method involves the step of culturing agene expression system which comprises a host cell which has beenrecombinantly modified with a polynucleotide encodingα1,3-fucosyltransferase or a portion thereof and harvesting theα1,3-fucosyltransferase. A preferred embodiment of the method isdirected to the use of the claimed genetic expression system whichproduces α1,3-fucosyltransferase.

[0011] These and many other features and attendant advantages of thepresent invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the Examples.

ABBREVIATIONS

[0012] The abbreviation used are: FucT, α1,3-fucosyltransferase unlessspecified otherwise; Le^(x), Lewis X; sLe^(x), sialyl-Lewis X; Le^(y),Lewis Y; nt, nucleotide (s); kb, kilobase (s); aa, amino acid (s); PCR,polymerase chain reaction; ORF, open reading frame; RSB, a ribosomalbinding site; LPS, lipopolysaccharides; HD-Zip, homeodomain-leucinezipper; bZip, basic region-zipper; LacNAc-R,Galβ1-4GlcNAcβ-O—(CH₂)₈COOMe;Galβ1-3GlcNAc—R,Galβ1-3GlcNAcb-O—(CH²)⁸COOMe; LacNAc-TMR,Galβ1-4GlcNAcβ-O—(CH₂)₈CO—NHCH₂CH₂NH-TMR; Phenyl-Gal,phenyl-β-galactoside.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Schematic representation of plasmid constructs containingan intact or partial HpfucT gene. Hatched arrow bars represent the H.pylori fucT genes, and the arrows point in the direction of thetranscription orientation. T7 indicates the location of a T7 promoter.Restriction endonuclease sites used for subcloning are denoted.BA=BamHI; B=BglII; X=XmnI; H=HindIII; and E=EcoRI.

[0014]FIG. 2A shows nucleotide (SEQ ID NO: 4) and deduced amino acid(SEQ ID NO: 1) sequences of H. pylori fucT gene. A Shine-Dalgarno (SD)sequence, the Kozak's consensus context, putative −10 and −35 regions,an asymmetric inverted repeat and a putative transcription terminatorare indicated in the nucleotide sequence. Putative asparagine-linkedglycosylation sites are underlined in the amino acid sequence. Primersused for construction of pBKHp763fucT38 and pBKHp763fucT39 are locatedby arrow bars.

[0015]FIG. 2B, a hydropathy profile of HpFucT as predicted by the methodof Kyte-Doolittle.

[0016]FIG. 3A, shows representative sequence alignment of the HpFucTwith eukaryotic α-1,3-fucosyltransferases using the program of Pileup(the GCG package, version 8.0). BfucT3=bovine FucT III; HfucT6=humanFucT VI; MfucT4=mouse FucT VI; CfucT1=chicken FucT1. Underlined residuesrepresent the proposed transmembrane segment within the respectiveFucTs. Identical residues within all the aligned proteins are denoted byboth asterisks and bold type: Corresponding residues partially conservedby HpFucT and other FucTs are indicated by bold type alone.

[0017]FIG. 3B, shows a sequence comparison of the direct repeat regionof HpFucT with the leucine zipper motifs within the chicken EAP-300protein, HD-Zip proteins, and bZip proteins. Conserved leucines amongall the compared proteins are marked by asterisks and bold type. Degreesof sequence identity and similarity (including the conservativereplacement) between HpFucT and ATHD-Zip proteins are given on the rightin panel B. ATHD-Zip, Aabidoposis thaliana homeobox-leucine zipperproteins; EAP-300 is a developmentally regulated embryonal protein;TAF-1, is a tobacco transcription activator 1; CPRF1, is a common plantregulatory factor isolated from parsley; TodS, is a histidine kinase inPseudomonas putida F1. Numbers in panels A and B indicate the alignedregions corresponding to the respective proteins.

[0018]FIG. 4 is a electrophoresis gel showing over expression of the H.pylori fucT gene in E. coli CSRDE3 cells. Equal amounts of the proteinextracts as determined by the turbidity of the cultures were separatedon a 13.5% polyacrylamide gel. Lane 1, pBKHp763fucT38; Lane 2,pBKHp763fucT39; Lane 3, pBluescript II KS-; Lane 4, no plasmid. Theproteins bands of interest and molecular mass makers (BRL/Gibco) wereindicated by arrow heads and lines on the left, respectively.

[0019]FIG. 5 shows graphical analysis of reaction mixtures containingthe membrane fraction of cells harboring pBKHp763fucT39 by capillaryelectrophoresis with laser-induced fluoresence detection. FIG. 5A is anelectropherogram showing the reaction product from an incubationcontaining LacNAc-TMR and GDP-fucose with the membrane extract frompBKHp763fucT39. Lewis X (Galβ1→4[Fucα1→3]GlcNAcβ-TMR) and GlcNAcβ-TMRwere formed and confirmed by both co-injection with standards andtreatment with a-fucosidase. FIG. 5B is an electropherogram showing thereaction mixture obtained from a-fucosidase treatment containingLewis-X-TMR with a 36% reduction in fluorescence signal. The GlcNAc-TMRpeak also had a corresponding increase in intensity by 39%. (c)Separation of nine standard TMR oligosaccharides found in mammalianmetabolism, LacNAcβ-(1), Fucαa1→2Galβ1→4GlcNAcβ-(2),Galβ1→4[Fucα1→3]GlcNAcβ-(3), Fucα1→2Galβ1→4[Fucα1→3]GlcNAcβ-(4),GlcNAcβ-(5), linker arm-(6), NeuAcα2→6LacNAcβ-(7), NeuAcα2→3LacNAcβ-(8),NeuAcα2→3Galβ1→4[Fucα1→3]GlcNAcβ-TMR(9).

[0020]FIG. 6 shows the amino acid sequence comparison among HpFuc-Tsfrom different H. pylori strains. HpFuc-Ts: 26695A/B from strain 26695;1182 from UA1182 (SEQ ID NO: 2); 763 from NCTC11639; 11637 fromNCTC11637; and 802 from UA802 (SEQ ID NO: 3). The position leading tothe frameshift is indicated by a ↑.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to novel purified alα1,3-fucosyltransferase polypeptides, polynucleotides which encode the aα1,3-fucosyltransferase, and the use of the α1,3-fucosyltransferase geneand a α1,3-fucosyltransferase polypeptides in the production ofbiologics and in the screening of biological tissues and fluids. Theinvention also relates to antibodies against α1,3-fucosyltransferasepolypeptides and their use in diagnosing disorders and in monitoringdisease.

[0022] The α1,3-fucosyltransferase Polypeptide

[0023] The amino acid sequence encoded by the α1,3-fucosyltransferasegene is shown in FIG. 2. In one embodiment, the α1,3-fucosyltransferaseis transmembrane segment-free. The term “transmembrane segment-free”refers the absence of a transmembrane segment found in eukaryoticα1,3-fucosyltransferase. Absence of a transmembrane segment allows theα1,3-fucosyltransferase of the invention to be readily released fromcells expressing the enzyme. Further, because theα1,3-fucosyltransferase are prokaryotically derived post-translationalmodifications are not made to the enzyme, unlike the eukaryoticallyexpressed α1,3-fucosyltransferase.

[0024] Additionally, the α1,3-fucosyltransferase polypeptide may bealtered by addition or deletions of peptide sequences in order to modifyits activity. For example, polypeptide sequences may be fused to theα1,3-fucosyltransferase polypeptide in order to effectuate additionalenzymatic activity. Alternatively, amino acids may be deleted to removeor modify the activity of the protein. The protein may be modified tolack α1,3-fucosyltransferase enzymatic activity but yet retain itsstructural three-dimensional structure. Such modification would beuseful in the development of antibodies against α1,3-fucosyltransferasepolypeptide as described more fully below.

[0025] Another embodiment relates to the direct repeats of seven aminoacid residues proximal to the C-terminus. These heptad repeats have thestructure: X₁X₂LRX₃X₄Y, wherein X₁ and X₂ are independently D or N; X₃is I, V or A; X₄ is N or D. The number of heptad repeats whichpotentially constitute a leucine zipper (L-Zip) may be varied (FIG. 6).Another embodiment is directed to the amino acid substitutionsintroduced into these heptad repeats.

[0026] In yet another embodiment, the invention includes aspects of theenzymatic activity of α1,3-fucosyltransferase, wherein theα1,3-fucosyltransferase polypeptide lacks α1,4-fucosyltransferase orα1,2-fucosyltransferase activity or lacks both α1,2-fucosyltransferaseand α1,4-fucosyltransferase activity.

[0027] The α1,3-fucosyltransferase gene product may include thoseproteins encoded by the α1,3-fucosyltransferase gene sequences describedin the section below. Specifically, α1,3-fucosyltransferase geneproducts, sometimes referred to herein as “α1,3-fucosyltransferasepolypeptides”, may include α1,3-fucosyltransferase gene product encodedby an α1,3-fucosyltransferase gene sequence shown in FIG. 2 and SEQ IDNO: 4. Thus, the term “α1,3-fucosyltransferase polypeptide” includesfull length expression as well as polypeptides, such as smallerpeptides, which retain a biological activity of the full length product,such as α1,3-fucosyltransferase activity.

[0028] In addition, α1,3-fucosyltransferase gene products may includeproteins or polypeptides that represent functionally equivalent geneproducts, for example and not by way of limitation, the sequences of SEQID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Such an equivalentα1,3-fucosyltransferase gene product may contain deletions, additions orsubstitutions of amino acid residues within the amino acid sequenceencoded by the α1,3-fucosyltransferase gene sequences described above,but which results in a silent change, thus producing a functionallyequivalent α1,3-fucosyltransferase gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

[0029] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; planar neutral amino acids include glycine,, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa polypeptide capable of exhibiting a substantially similar in vivoactivity as the endogenous α1,3-fucosyltransferase gene products encodedby the α1,3-fucosyltransferase gene sequences described above, as judgedby any of a number of criteria, including but not limited toantigenicity, i.e., the ability to bind to ananti-α1,3-fucosyltransferase antibody, immunogenicity, i.e., the abilityto generate an antibody which is capable of binding aα1,3-fucosyltransferase protein or polypeptide, as well as enzymaticactivity.

[0030] A substantially purified α1,3-fucosyltransferase protein,polypeptide, and derivative (including a fragment) is substantially freeof other proteins, lipids, carbohydrates, nucleic acids, and otherbiological materials with which it is naturally associated. For example,a substantially purified functional fragments of α1,3-fucosyltransferasepolypeptide can be at least 60%, by dry weight, the molecule ofinterest. One skilled in the art can purify functional fragment ofα1,3-fucosyltransferase protein using standard protein purificationmethods and the purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography), and amino-terminal amino acid sequence analysis.

[0031] Included within the scope of the invention areα1,3-fucosyltransferase proteins, polypeptides, and derivatives(including fragments) which are differentially modified during or aftertranslation. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc. Additionally,the composition of the invention may be conjugated to other molecules toincrease their water-solubility (e.g., polyethylene glycol), half-life,or ability to bind targeted tissue.

[0032] Furthermore, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into theα1,3-fucosyltransferase polypeptide sequence. Non-classical amino acidsinclude, but are not limited to, the D-isomer of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,γ-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids, such as β-methyl amino acids,α-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

[0033] While random mutations can be made to α1,3-fucosyltransferase DNA(using random mutagenesis techniques known to those skilled in the art)and the resulting mutant α1,3-fucosyltransferase polypeptides tested foractivity, site-directed mutation of the α1,3-fucosyltransferase codingsequence can be engineered (using site-directed mutagenesis techniqueswell known to those skilled in the art) to create mutantα1,3-fucosyltransferase polypeptides with increased functionalcharacteristics.

[0034] Peptides corresponding to one or more domains of theα1,3-fucosyltransferase protein, truncated or deletedα1,3-fucosyltransferase proteins as well as fusion proteins in which thefull length α1,3-fucosyltransferase proteins, polypeptides orderivatives (including fragments), or truncated α1,3-fucosyltransferase,is fused to an unrelated protein are also within the scope of theinvention and can be designed on the basis of theα1,3-fucosyltransferase nucleotide and α1,3-fucosyltransferase aminoacid sequences disclosed in this section and the section above. Thefusion protein may also be engineered to contain a cleavage site locatedbetween a α1,3-fucosyltransferase sequence and thenon-α1,3-fucosyltransferase protein sequence, so that theα1,3-fucosyltransferase polypeptide may be cleaved away from thenon-α1,3-fucosyltransferase moiety. Such fusion proteins or polypeptidesinclude but are not limited to IgFc fusion which may stabilize theα1,3-fucosyltransferase protein in vivo; or fusion to an enzyme,fluorescent protein, or luminescent protein which provide a markerfunction.

[0035] The α1,3-fucosyltransferase polypeptide may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the α1,3-fucosyltransferase polypeptides of theinvention by expressing nucleic acid containing α1,3-fucosyltransferasegene sequences are described herein. Method which are well known tothose skilled in the art can be used to construct expression vectorscontaining α1,3-fucosyltransferase coding sequences and appropriatetranscriptional translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniques,and in vivo genetic recombination. See, for example, the techniquesdescribed in Sambrook et al., 1989, supra, and Ausubel et al., 1989,supra. Alternatively, RNA capable of encoding α1,3-fucosyltransferasepolypeptide may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety. The use ofsuch synthetic peptide fragments of α1,3-fucosyltransferase forgenerating polyclonal antibodies is described below.

[0036] The α1,3-fucosyltransferase Gene

[0037] The α1,3-fucosyltransferase gene is a novel gene (FIG. 2) whoseexpression is found in H. pylori. Nucleic acid sequences of theidentified α1,3-fucosyltransferase genes are described herein. As usedherein, “α1,3-fucosyltransferase gene” refers to (a) a gene containingthe DNA sequence shown in FIG. 2; (b) any DNA sequence that encodes theamino acid sequence shown in FIG. 2, SEQ ID NO: 1, SEQ ID NO: 2 or SEQID NO: 3; (c) any DNA sequence that hybridizes to the complement of thecoding sequences shown in FIG. 2, SEQ ID NO: 1, SEQ ID NO: 2 or SEQ IDNO: 3 under stringent conditions, e.g., hybridization to filter-boundDNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al.,eds., 1989, Current Protocols in Molecular Biology, Vol. 1, GreenPublishing Associates, Inc., and John Willey & Sons, Inc., New York, atp. 2.10.3) and encodes a gene product functionally equivalent to a geneproduct encoded by sequences shown in FIG. 2; and/or (d) any DNAsequence that hybridizes to the complement of the coding sequencesdisclosed herein (as shown in FIG. 2), under less stringent conditions,such as moderately stringent conditions, e.g., washing in,0.2% SSC/0.1%SDS at 42° C. (Ausubel et al., 1989, supra), and encodes a gene productfunctionally equivalent to a gene product encoded by sequences shown inFIG. 2.

[0038] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the DNA sequences (a) through (c), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentcondition may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act at α1,3-fucosyltransferase generegulation and/or as antisense primers in amplification reactions ofα1,3-fucosyltransferase gene nucleic acid sequences. Further, suchsequences may be used as part of ribozyme and/or triple helix sequences,also useful for α1,3-fucosyltransferase gene regulation. Still further,such molecules may be used as components of diagnostic methods wherebythe presence of a pathogen or metastatic tumor cell may be detected.

[0039] The invention also encompasses (a) DNA vectors that contain anyof the foregoing coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingcoding sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

[0040] The invention includes fragments of any of the DNA sequencesdisclosed herein. Fragments of the α1,3-fucosyltransferase genecorresponding to coding regions of particular domains, or in which oneor more of the coding regions of the domains is deleted (e.g., thesequence encoding the c-terminal 101 amino acids as shown in FIG. 2),are especially useful. Such α1,3-fucosyltransferase gene fragments mayencode truncated gene products that retain a biological activity of thefull-length α1,3-fucosyltransferase polypeptide, such asα1,3-fucosyltransferase activity or immunogenicity. The invention alsoincludes mutant α1,3-fucosyltransferase genes encoding substitutions ofamino acids as described below.

[0041] In addition to the gene sequences described above, homologs ofsuch sequences, as may, for example, be present in other species,including humans, may be identified and may be readily isolated, withoutundue experimentation, by molecular biological techniques well known inthe art. Further, there may exist genes at other genetic loci within thegenome that encode proteins which have extensive homology to one or moredomains of such gene products. These genes may also be identified viasimilar techniques.

[0042] The α1,3-fucosyltransferase gene and its homologs can be obtainedfrom other organisms thought to contain α1,3-fucosyltransferaseactivity. For obtaining cDNA, tissues and cells in whichα1,3-fucosyltransferase is expressed are optimal. Tissues which canprovide a source of genetic material for α1,3-fucosyltransferase and itshomologs, therefore, include intestinal mucosal cells and tumorigeniccells.

[0043] For example, the isolated α1,3-fucosyltransferase gene sequencesmay be labeled and used to screen a cDNA library constructed from mRNAobtained from the organism of interest. The hybridization conditionsused should be of a lower stringency when the cDNA library is derivedfrom an organism different from the type of organisms from which thelabeled sequence was derived. Alternatively, the labeled fragment may beused to screen a genomic library derived from the organism of interest,again, using appropriately stringent condition. Low stringencyconditions are well known in the art, and will vary predictablydepending on the specific organism from which the library and thelabeled sequences are derived. For guidance regarding such conditionsee, for example, Sambrook et al., 1989, Molecular Cloning, a LaboratoryManual, Cold Springs Harbor Press, N.Y; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y

[0044] Further, a previously unknown α1,3-fucosyltransferase gene typesequence may be isolated by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequence within the gene of interest. The template for the reaction maybe cDNA obtained by reverse transcription of mRNA prepared from human ornon-human cell lines or tissue known or suspected to express aα1,3-fucosyltransferase gene.

[0045] The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a α1,3-fucosyltransferasegene-like nucleic acids sequences. The PCR fragment may then be used toisolate a full length cDNA clone by a variety of methods. For example,the amplified fragment may be labeled and used to screen a bacteriophagecDNA library. Alternatively, the labeled fragment may be used to screena genomic library.

[0046] PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanidines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

[0047] In cases where the α1,3-fucosyltransferase gene identified is thenormal, or wild type, gene, this gene may be used to isolate mutantalleles of the gene. Mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes tointestinal mucosal disease and/or tumorigenicity. Mutant alleles andmutant allele products may then be utilized in the therapeutic anddiagnostic systems described below.

[0048] A cDNA of the mutant gene may be isolated, for example by PCR. Inthis case, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically the 5′ end of the normal gene. Using theseprimers, the product is then amplified via PCR, cloned into a suitablevector, and subjected to DNA sequences analysis through methods known inthe art. By comparing the DNA sequence of the mutant gene to that of thenormal gene, the mutation(s) responsible for the loss or alteration offunction of the mutant gene product can be ascertained.

[0049] A variety of host-expression vector systems may be utilized toexpress the α1,3-fucosyltransferase gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit theα1,3-fucosyltransferase gene product of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containingα1,3-fucosyltransferase gene product coding sequences; yeast (e.g.Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the α1,3-fucosyltransferase gene product codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing theα1,3-fucosyltransferase gene product coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing α1,3-fucosyltransferase gene product codingsequences; or mammalian cell systems (e.g., COS, SHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0050] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for theα1,3-fucosyltransferase gene product being expressed. For example, whena large quantity of such a protein is to be produced, for the generationof pharmaceutical compositions of α1,3-fucosyltransferase polypeptide orfor raising antibodies to α1,3-fucosyltransferase polypeptide, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which theα1,3-fucosyltransferase gene product coding sequence may be ligatedindividually into the vector in frame with the lac z coding region thata fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109); and the like. pGEX vectors may also beused to express foreign polypeptide as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0051] In an insect system, Autographa colifornica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperday cells. The α1,3-fucosyltransferase genecoding sequence may be cloned individually into non-essential regions(for example the polyhedrin gene) of the virus and placed under thecontrol of an AcNPV promoter. Successful insertion ofα1,3-fucosyltransferase gene coding sequence will result in inactivationof the polyhedrin gene and production of non-occluded recombinant virus.These recombinant viruses are then used to infect S. frugiperda cells inwhich the inserted gene is expressed.

[0052] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the α1,3-fucosyltransferase gene coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing α1,3-fucosyltransferasegene product in infected hosts (See Logan & Shenk, 1984, Proc. Natl.Acad. Sci, USA 81:3655-3659). Specific initiation signals may also berequired for efficient translation of inserted α1,3-fucosyltransferasegene product coding sequences. These signals include the ATG initiationcodon and adjacent sequences. In cases where an entireα1,3-fucosyltransferase gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translation control signals may be needed. However, incases where only a portion of the α1,3-fucosyltransferase gene codingsequences is inserted, exogenous translational control signals,including, the ATG initiation codon must be provided.

[0053] Transfection via retroviral vectors, naked DNA methods andmechanical methods including micro injection and electroporation may beused to provide either stably transfected host cells (i.e., host cellsthat do not lose the exogenous DNA over time) or transient transfectedhost cells (i.e., host cells that lose the exogenous DNA during cellreplication and growth).

[0054] An alternative fusion protein system allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cell infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0055] The α1,3-fucosyltransferase gene products can also be expressedin transgenic animals. Animals of any species, including, but notlimited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats,and non-human primates may be used to generate α1,3-fucosyltransferasetransgenic animals.

[0056] Expression Systems for α1,3-fucosyltransferase

[0057] The novel bacterial α1,3-fucosyltransferase encoded by thedisclosed gene, and enzymatically active fragment thereof, can be usedin the production of fucosylated oligosaccharides such as Lewis X, LewisY, and siayl Lewis X. These bacterial oligosaccharides are structurallysimilar to certain tumor-associated carbohydrate antigens found inmammals. These product glycoconjugates also have research and diagnosticutility in the development of assays to detect mammalian tumors.

[0058] The fucosylated oligosaccharides may be produced by any number ofmethods utilizing the methods and compositions described herein.Standard enzymology techniques well known in the art may be utilized todevelop systems to provide fucosylated oligosaccharides (see for examplethe Methods in Enzymology, volume series published by Academic Press;and Tim Bugg, “An Introduction to Enzyme and Coenzyme Chemistry”, 1997,Blackwell Sciences, Inc.).

[0059] “Substrate”, as used herein, means any material or combinationsof different materials, that may be acted upon by the polypeptide of theinvention to give rise to fucosylated oligosaccharides, for example, andnot by way of limitation, substrates may include LacNAc-R andGDP-fucose.

[0060] Cells containing and cell-free systems may be used to produce thefucosylated oligosaccharides of the present invention. Cells containingand cell-free systems will be better understood in the description andexamples that follow. Such systems are useful in the development offucosylated oligosaccharides.

[0061] The present invention provides a method for synthesizingfucosylated oligosaccharides by reacting substrates in the presence ofα1,3-fucosyltransferase, capable of catalyzing the formation of thefucosylated oligosaccharides from the substrates.

[0062] The α1,3-fucosyltransferase may be used regardless of its originas long as it is capable of producing the fucosylated oligosaccharidesfrom the substrates. The source of the α1,3-fucosyltransferase may bederived according to the methods and compositions as described herein,for example, through protein purification from host cells transfectedwith an expression system as described more fully below.

[0063] The substrates are allowed to react with theα1,3-fucosyltransferase polypeptide for a sufficient time and undersufficient conditions to allow formation of the enzymatic product, e.g.Le^(x), Le^(y) and sLe^(x). These conditions will vary depending uponthe amounts and purity of the substrate and enzyme, whether the systemis a cell-free or cellular based system. These variables will be easilyadjusted by those skilled in the art. For example, the period ofexposure of the enzyme to the substrate will be longer at lowertemperatures, e.g., 4° C. rather than at higher temperatures. In themethods for synthesizing the fucosylated oligosaccharides there are norestriction in terms of the timing of the addition of the substrates.The ratios of the various substrates should be in equal proportions,i.e. 1:1. The ratios of the enzyme to the substrates may be varieddepending upon the rate and quantity of fucosylated oligosaccharidesdesired.

[0064] The method of producing the fucosylated oligosaccharides may becarried out at temperatures of 4° C. to 60° C., more specifically at 20°C. to 45° C. Additionally, a number of buffers may be used, for example,and not by way of limitation, a buffer having a pH between 6.5 and 8.0,but more preferably at pH 7.5, and in the presence of 15-30 mM Mn²⁺ butmore preferably at a 25 mM Mn²⁺ concentration. After a desired amount offucosylated oligosaccharides are produced the α1,3-fucosyltransferasepolypeptide may be inactivated by heating, centrifugal separation, orthe like. The resulting fucosylated oligosaccharides may be furtherpurified by techniques known to those skilled in the art.

[0065] Cell containing systems for the synthesis of fucosylatedoligosaccharides may include recombinantly modified host cells accordingto the methods described below or may be naturally occurring cells whichexpress α1,3-fucosyltransferase polypeptide or an enzymatically activeportion thereof, so long as the cell is capable of catalyzing thesynthesis of fucosylated oligosaccharides from substrates.

[0066] In the case of cell containing systems the host cell is contactedwith the substrate, under conditions and for sufficient time to producethe oligosaccharide. The time and conditions will vary depending uponthe host cell type and culture conditions and can be easily determinedby those of skill in the art.

[0067] The invention provides a gene expression system for producingα1,3-fucosyltransferase polypeptides. The gene expression systemcomprises a host cell which been modified with a polynucleotide encodingα1,3-fucosyltransferase polypeptide or a portion thereof, as describedabove.

[0068] A preferred gene expression system of the invention involves hostcell modified with a polynucleotide encoding α1,3-fucosyltransferasepolypeptide or a portion thereof.

[0069] The method involves culturing a gene expression system createdaccording to the methods described above under conditions sufficient toproduce the α1,3-fucosyltransferase polypeptide. The gene expressionsystem comprises a host cell which has been recombinantly modified witha polynucleotide encoding a α1,3-fucosyltransferase polypeptide or aportion thereof.

[0070] The method is also directed to harvesting theα1,3-fucosyltransferase polypeptide. A further step of the methodinvolves substantially purifying the harvested α1,3-fucosyltransferase.The purified α1,3-fucosyltransferase polypeptide may be used in thesynthesis of fucosylated oligosaccharides or the preparation ofantibodies as described above.

[0071] Specifically disclosed herein is a gene expression systemrecombinantly modified with a DNA sequence containing theα1,3-fucosyltransferase gene. The sequence contains an open readingframe (ORF) of approximately 1211 base pairs which are transcribed intoα1,3-fucosyltransferase product.

[0072] As used herein, the term “recombinantly modified” meansintroducing a polynucleotide encoding α1,3-fucosyltransferasepolypeptide into a living cell or gene expression system. Usually, thepolynucleotide is present in a plasmid or other vector, althoughmodification can also occur by uptake of free α1,3-fucosyltransferasepolynucleotide or numerous other techniques known in the art.

[0073] As used herein, the term “gene expression system” means a livingeukaryotic or prokaryotic cell into which a gene, whose product is to beexpressed, has been introduced, as described above.

[0074] As used herein, the term “harvesting” means collecting orseparating from the gene expression system the product produced by theinserted polynucleotide.

[0075] Polynucleotide sequences encoding α1,3-fucosyltransferasepolypeptides can be expressed by polynucleotide transfer into a suitablehost cell.

[0076] “Host cells” are cells in which a vector can be propagated andits DNA expressed. A gene expression system is comprised of a host cellin which a vector was propagated and the vector's DNA expressed. Theterm “host cell” also includes any progeny of the subject host cell. Itis understood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. However,such progeny are included when the term “host cell” is used. Host cellswhich are useful in the claimed gene expression system and the claimedmethod of producing α1,3-fucosyltransferase polypeptide includebacterial cells, yeast cells fungal cells, plant cells and animal cells.

[0077] Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art. In thepresent invention, the α1,3-fucosyltransferase polynucleotide sequencesmay be inserted into a recombinant expression vector. The term“recombiant expression vector” refers to a plasmid, virus or othervehicle known in the art that has been manipulated by insertion orincorporation of the α1,3-fucosyltransferase genetic sequences. Suchexpression vectors contain a promoter sequence which facilitates theefficient transcription of the inserted genetic sequence of the host.The expression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart. Such vectors are used to incorporate DNA sequences of theinvention.

[0078] The method of the invention produces α1,3-fucosyltransferasepolypeptide which are substantially pure. As used herein, the term“substantially pure” refers to a protein which is free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated. One skilled in the art can purifyα1,3-fucosyltransferase polypeptide using standard techniques forprotein purification including preparative chromatography andimmunological separations involving monoclinal or polyclonal antibodies.For example, the substantially pure α1,3-fucosyltransferase protein willyield a single major band of approximately 52 kD on a non-reducingpolyacrylamide gel. The purity of the α1,3-fucosyltransferasepolypeptide can also be determined by amino-terminal amino acid sequenceanalysis. α1,3-fucosyltransferase polypeptide include functionalfragments of the polypeptide, as long as biological activity remains,such as α1,3-fucosyltransferase enzymatic activity. Accordingly, theinvention includes a gene expression system and a method of producingα1,3-fucosyltransferase polypeptide which produce smaller peptidescontaining the enzymatic activity of α1,3 -fucosyltransferase.

[0079] Production of α1,3-fucosyltransferase. Production ofα1,3-fucosyltransferase from the gene expression system of the inventionis achieved by culturing a gene expression system comprising a host cellrecombinantly modified with a polynucleotide encodingα1,3-fucosyltransferase polypeptide or an enzymatically active portionthereof and harvesting the α1,3-fucosyltransferase polypeptide. Themethod further comprises substantially purifying the harvestedα1,3-fucosyltransferase polypeptide using protein purification protocolswell known in the art (Current Protocols in Molecular Biology, Chapter10, eds. Ausubel, F. M. et al., 1994).

[0080] The method for producing α1,3-fucosyltransferase polypeptideinvolves culturing the gene expression system of the invention underconditions of continuous culture, such as, but not restricted to,“fed-batch cultures” or continuous perfusion cultures. Other continuousculture systems which find use in the present invention is set forth inWang, G. et al. Cytotechnology 9:41-49, 1992; Kadouri, A. et al.Advances in Animal Cell Biology and Technology for Bioprocesses, pp.327-330, Courier International, Ltd., 1989; Spier, R. E. et al.Biotechnol. Bioeng. 18:649-57, 1976. TABLE 1 Enzyme activity of the H.pylori FucT produced in E. coli CSRDE3 cells with an acceptor LacNAc-RActivity Specific Relative Sample^(a) (mU)^(b) activity^(c) activity^(d)BKHp763fucT38 cytoplasm 0 0  0 membrane 0 0  0 pBKHp763fucT39 cytoplasm0.6 0.026 15% membrane 3.4 0.62 85% membrane + 4.3 0.77 — Triton X-100

[0081] Antibodies to α1,3-fucosyltransferase Proteins

[0082] Antibodies that define the α1,3-fucosyltransferase gene productare within the scope of this invention, and include antibodies capableof specifically recognizing one or more α1,3-fucosyltransferase geneproduct epitopes. Such antibodies may include, but are not limited to,polyclonal antibodies, monoclinal antibodies, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection ofα1,3-fucosyltransferase gene product in a biological sample, including,but not limited to, blood, plasma, and serum. Alternatively, theantibodies may be used as a method for the inhibition of abnormalα1,3-fucosyltransferase gene product activity. Thus, such antibodies maybe utilized as part of treatment for intestinal mucosal disease, and maybe used as part of diagnostic techniques whereby patients may be testedfor abnormal levels of α1,3-fucosyltransferase gene products, or for thepresence of abnormal forms of such proteins.

[0083] For the production of antibodies against aα1,3-fucosyltransferase gene product, various host animals may beimmunized by injection with a α1,3-fucosyltransferase gene product, or aportion thereof. Such host animals may include but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsion, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG, interferon and other cytokineseffecting immunological response.

[0084] Polyclonal antibodies are a heterogenous population of antibodymolecules derived from the sera of animals immunized with an antigen,such as a α1,3-fucosyltransferase gene product, or an antigenicfunctional derivative thereof. In general, for the production ofpolyclonal antibodies, host animals such as those described above, maybe immunized by injection with α1,3-fucosyltransferase gene productsupplemented with adjuvants as also described above.

[0085] Monoclonal antibodies (mAbs), which are homogenous population ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These techniques include, but are not limited to,the hybridoma technique of Kohler and Milstein, (1975, Nature256:495-497; and U.S. Pat. No. 4,376,110), human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclinal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

[0086] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0087] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against α1,3-fucosyltransferase geneproducts. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

[0088] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclinal Fab fragments with thedesired specificity.

[0089] Methods of Detecting α1,3-fucosyltransferase in BiologicalSamples

[0090] The antibodies described above can be used in the detection ofα1,3-fucosyltransferase polypeptides in biological samples.α1,3-Fucosyltransferase polypeptide from blood or other tissue or celltype may be easily isolated using techniques which are well known tothose of skill in the art. The protein isolation methods employed hereinmay, for example, be such as those described in Harlow and Lane (Harlow,E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety.

[0091] Preferred diagnostic method for the detection of wild type ormutant α1,3-fucosyltransferase polypeptides may involve, for example,immunoassays wherein α1,3-fucosyltransferase polypeptides are detectedby their interaction with an anti-α1,3-fucosyltransferase polypeptidespecific antibody.

[0092] For example, antibodies, or fragments of antibodies, such asthose described above, useful in the present invention may be used toquantitatively or qualitatively detect the presence of wild type ormutant α1,3-fucosyltransferase polypeptides. This can be accomplished,for example, by immunofluorescence techniques employing a fluorescentlylabeled antibody coupled with light microscopic, flow cytometric, orfluorimetric detection. Such techniques are especially preferred if theα1,3-fucosyltransferase polypeptides are expressed on the cell surface.

[0093] The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof α1,3-fucosyltransferase polypeptides. In situ detection may beaccomplished by removing a histological specimen from a patient, andapplying thereto a labeled antibody of the present invention. Theantibody (or fragment) is preferably applied by overlaying the labeledantibody (or fragment) onto a biological sample. Through the use of sucha procedure, it is possible to determine not only the presence of theα1,3-fucosyltransferase polypeptide, but also its distribution in theexamined tissue. Using the present invention, those skill in the artwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

[0094] Immunoassays for wild type or mutant α1,3-fucosyltransferasepolypeptides typically comprise incubating a biological sample, such asa biological fluid, including but not limited to blood, plasma, or bloodserum, a tissue extract, freshly harvested cells, or cells which havebeen incubate in tissue culture, in the presence of a detectably labeledantibody capable of identifying α1,3-fucosyltransferase polypeptides,and detecting the bound antibody by any of a number of techniques wellknown in the art.

[0095] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling the antibodyor antibody fragments, it is possible to detect wild type or mutantα1,3-fucosyltransferase polypeptides through the use ofradioimmunoassays (RIA) (see, for example, Weintraub, Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography.

[0096] It is also possible to label the antibody with a fluorescentcompound such fluorescein isothiocyanate, rhodomine, phycoerythrin,phycocyanin, allophycocyanin and fluorescamine.

[0097] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu. Additionally the antibody may be detectedby coupling it to a chemiluminescent compound such as luminol,isoluminol, theramatic acreidinium ester and oxalate ester.

[0098] The following examples are intended to illustrate but not limitthe invention. While they are typical, other procedures known to thoseskilled in the art may alternatively be used to illustrate theembodiments and methods of the invention.

EXAMPLE 1

[0099] Cloning of the H. pylori fucosyltransferase (fucT) gene To clonethe fucosyltransferase gene from H. pylori NCTC11639, degenerate primerswere generated from the several regions conserved by three mammalianα1-3 fucosyltransferases, including human FucT VI, bovine FucT III andmouse FucT VI. Primer FUTF3(5′TT[T/C]TA[TC]CT[T/C/A/G]GC[G/A/T/C]TT[T/C]GA[A/G]AA3′) corresponds toresidues 242-248 of human FucT VI, whereas primer FUCTR2(5′AA[A/G]TC[A/G]TC[G/ATC]AC[A/G]TG[G/A/T/C]AG[A/G]AA3′) iscomplementary to the sequence deduced from its residues 289-295. Anexpected DNA fragment of ˜170 nt was PCR-amplified from chromosomal DNAof H. pylori NCTC11639 with the primer pair of FUCTF3 and FUCTR2 under athermocycling program of 40 cycles: for the first two cycles, 1 min at94° C., 30 sec at 40° C. and 40 sec at 72° C.; for the remaining cycles,1 min. at 94° C., 30 sec at 50° C. and 40 sec at 72° C., followed byextension at 72° C. for 10 min. The PCR products were cloned into vectorpCRTMII (Invitrogen, San Diego, Calif.) according to the supplier'sinstructions. Subsequently, the inserts in recombinant plasmids weresequenced with Thermo sequenase, and their nucleotide sequences anddeduced amino acid sequences were used in the search for relatedproteins in databases with the software program Blast included in theGCG package (Version 8.0, Genetic Computing Group, Inc., Madison, Wis.).A clone, designated pCRHpfucT3, was demonstrated to contain the insertencoding the amino acid sequence homologous to known mammalianα1,3-fucosyltransferases.

[0100] To clone a putative intact fucT gene from H. pylori, chromosomalDNA from H. pylori NCTC11639 was digested with restrictionendonucleases, including Bg/II, EcoRI, BamHI, Bg/II-EcoRI, EcoRI-BamHIand Bg/II-BamHI, and then separated in a 1% agarose gel. DNA fragmentscontaining the putative fucT gene were demonstrated by Southernhybridization with a [α-³²P]dCTP-labeled probe made from pCRHpfucT3 DNA.The 2.2-kb EcoRI-Bg/II and 4.5-kb EcoRI-BamHI fragments were cloned intovector pBluescript II KS-(Stratagene, La Jolla, Calif.) which wasdigested with EcoRI and BamHI. Two clones, pBKHpfucT8 carrying a 2.2-kbEcoRI-BgIII fragment and pBKHpfucT31 carrying a 4.5 kb EcoRI-BamHI, wereselected for further characterization.

EXAMPLE 2

[0101] Plasmid constructs and Expression of the H. pylori fucT gene Toconstruct recombinant plasmids containing an intact or partial H. pylorifucT gene, three primers were generated from the nucleotide sequence inFIG. 1: ZGE37 corresponding to nucleotides 1-19; ZGE38 and ZGE39complementary to nucleotides 1215-1233 and 1660-1679 respectively. ZGE37contained a BamHI site, whereas ZGE38 and ZGE39 contained an EcoRI site.PCR products were amplified from pBKHpfucT31 with a primer pair ofeither ZGE37/ZGE38 or ZGE37/ZGE39. These PCR-amplified DNA fragmentswere digested with EcoRI and BamHI, and then cloned into pBluescript IIKS-(Stratagene, La Jolla, Calif.). The respective clones containing theH. pylori fucT gene of interest were screened by PCR with thecorresponding pair of the above primers. Two clones, designatedpBKHp763fucT38 and pBKHp763fucT39, contained a partial and an intact H.pylori fucT gene respectively. The coding region of the H. pylori fucTgene was controlled under the T7 promoter. The sequence of thePCR-amplified DNA fragments in pBKHp763fucT38 and pBKHp763fucT39 wasdetermined and demonstrated to be identical to that of the nativetemplate.

[0102] Recombinant plasmids pBKHp763fucT38 and pBKHp763fucT39 wereintroduced into E. coli CSRDE3 cells by electroporation. The proteinsencoded by the recombinant plasmids were over expressed in the presenceof either [³⁵S]methionine or cold methionine. The cells expressing[³⁵S]methionine-labeled proteins were boiled for 5 min in 1× samplebuffer and separated in a 13.5% SDS-polyacrylamide gel.

EXAMPLE 3

[0103] Fucosyltransferase assays E. coli CSRDE3 cells expressingnonradioactively labeled proteins encoded by pBKHp763fucT38 andpBKHp763fucT39 were harvested and suspended in Hepes buffer (20 mMHepes, pH 7.0) supplemented with 0.5 mM of phenylmethylsulfonic fluoride(a proteinase inhibitor). Subsequently, membrane and soluble fractionsof the cells were prepared after disruption with a French press. Themembrane pellets were resuspended in the same Hepes buffer, frozen inliquid nitrogen, and stored at −70° C. until use.

[0104] Assays of H. pylori α1,3 and α1,4 fucosyltransferase activitieswere conducted at 37° C. for 20 min in a volume of 20 μl containingeither 720 μM LacNAc-R for α1,3-fucosyltransferase activity,Galβ1-3GlcNAc-R for α1,4-fucosyltransferase activity, or 5.33 mMPhenyl-Gal for α1,2-fucosyltransferase activity, 50 μM GDP-fucose,100,000 d.p.m GDP-[³H]fucose, 20 mM Hepes buffer (pH 7.0), 20 mM MnCl₂,0.2% BSA and 8.5 μl of the enzyme fraction. The incubation mixtures wereloaded onto Sep-Pak Plus C-18 cartridges and the unreacted donor wasremoved by washing the cartridges with water. The reaction products wereeluted from the cartridges with 4 ml of methanol, and radioactivity wascounted in 10 ml of Ecolite (+) cocktail in a Beckman LS 1801scintillation counter (Palcic, M. M., Venot, A. P., Ratcliffe, R. M.,and Hindsaul, O. (1988) Carbohydr. Res. 190, 1-11).

[0105] Capillary electrophoresis assay The incubation mixtures contained16 μl of the membrane fraction containing the intact HpFucT protein, 100μM LacNAc-TMR, 100 μM GDP-fucose in a total volume of 20 μl of 20 mMHepes (pH 7.0) containing 20 mM MnCl2 and 0.2% BSA. Incubation was doneat 37° C. for 30 minutes. Subsequently, the sample was prepared andanalyzed by capillary electrophoresis by injecting 12 μl onto anelectrophoresis column (60 cm long) at 1 kV for 5 s. The electrophoreticseparations were performed at a running voltage of 400 V/cm.α-Fucosidase treatment was done by incubating the sample (10 mM totalTMR) with 4 mU almond meal α-fucosidase (Sigma-Aldrich, Canada, Ltd,Misissauga, Ontario) in a total volume of 40 μl of 50 mM sodium citratebuffer, pH 5.0 at 37° C. for 90 hours. Products were isolated andanalyzed by capillary electrophoresis as described above.

[0106] Results

[0107] Cloning and Nucleotide sequence of a H. pylori FucosyltransferaseGene Three recombinant plasmids, pCRHpfucT3, pBKHpfucT8 and pBKHpfucT31(FIG. 1) containing the intact or partial sequence of the fucT gene fromH. pylori NCTC11639, were obtained as described in the examples above.The nucleotide sequences of these recombinant clones were sequenced fromboth strands using nested primers. FIG. 2A shows a nucleotide sequenceof 1670 bp derived from clone pBKHpfucT31. The sequence is characterizedby a major open reading frame (ORF), starting at nucleotide 145 andending at nucleotide 1356, which was predicted in this region. As shownin FIG. 2A, an unusual sequence feature of this ORF was eight directrepeats of 21 nucleotides. An AA to GG transition at positions 12 and 13of this repeat has occurred in repeat copies III and VI. An SD sequence,a ribosomal binding site (RSB) in prokaryotes (Shine, J., and Dalgarno,L. (1974) Proc. Natl. Acad. Sci. USA 71, 1342-1346), precedes thepredicted translation initiation codon AUG. In addition, the sequence“ACCATGT”, which is similar to the Kozak's consensus context “ACCATGG”(a common RSB in eukaryotes) (Kozak, M. (1986) Cell 44, 229-292), alsoexists at the beginning of the ORF. Putative transcription elementsincluding −10 and −35 regions immediately upstream of the ORF, and astem-loop structure following the stop codon of the ORF, which probablyact as a transcription promoter and rho-independent transcriptionterminator (Platt, T. (1986) Annu. Rev. Biochem. 55, 339-372), wereidentified. An asymmetric inverted repeat sequence was found,encompassing 18 nucleotides and containing the putative −10 region.Another ORF downstream from the major ORF, in the opposite orientationas indicated in FIG. 2A, encodes the amino acid sequence similar to thecorresponding region of the glutamate dehydrogenase identified inCorynebacterium glutamicum (nucleotide sequence accession #S32227).

[0108] Features of the deduced amino acid sequence of the H. pylori fucTA protein consisting of 464 amino acids with a calculated molecular massof 54,429 daltons was predicted from this ORF. A hydropathy profile(FIG. 2B) which was calculated by the method of Kyte and Doolittle((1982) J. Mol. Biol. 157, 105-132), indicates that the deduced aminoacid sequence is primarily hydrophilic, and does not contain a potentialtransmembrane segment (“transmembrane segment-free”).

[0109] Additionally, the predicted protein from the NTCT11639 H. pyloristrain carries eight direct repeats of seven amino acid residuesproximal to the C-terminus. There is a conservative replacement ofvaline by isoleucine at position 5 found in repeats III and VI, whichresults from the corresponding AA to GG mutations as mentioned above.Cloning of a number HpFucT from additional strains of H. pylori havedemonstrated that the amino acid sequence is highly conserved (77%identity and 87% similarity) except for the heptad repeats. H. pyloricontains two copies of HpFucT. The number of heptad repeats whichpotentially constitute a leucine zipper (L-Zip) domain are highlyvariable among the HpFucTs cloned and range from 11 to 3 in the variousstrains of H. pylori. (FIG. 6) There are some substitutions introducedinto the heptad repeats. The repeat unit in UA1182 H. pylori strain is“DDLRVNY”, whereas in the UA802 strain the repeat unit is “NNLRADY”.(FIG. 6) Searches for sequence similarly shown in FIG. 3B revealed thatthis region of repeats in HpFucT is significantly similar to domainspotentially forming a leucine-zipper structure within severalhomeobox-leucine zipper proteins (HD-Zip protein) including ATHB-1,ATHB-5 to 7 from Arabidoposis thaliana and tomato (nucleotide sequenceaccession #x94947) (FIG. 3B). The conserved leucine residues are alsocolinear to those present in the leucine zipper motif found in a groupof basic region -leucine zipper (bZip) proteins in eukaryotes, includingyeast, higher plant, animals, and recently, in a bacterium.

[0110] Five putative N-linked glycosylation sites are predicted, two ofwhich are proximal to the N-terminus, similar to those identified inmammalian FucTs. However, the remaining three such sites are close tothe C-terminus. This latter feature is similar to the sites identifiedin rabbit and human α1,2-fucosyltransferases (Hitoshi, S., Kusunoki, S.,Kanazawa, I., and Tsuji, S. (1996) J. Biol. Chem. 264, 17615-17618).Comparison of this polypeptide sequence with other proteins in theprotein data bases, using the Blast search program (Version 8.0, GeneticComputing Group, Inc., Madison, Wis.) revealed significant sequencesimilarity (40-45% identity) to α1,3 and 1-3/1-4 fucosyltransferasesfrom mammalian sources, including human FucT III to VII, bovine FucT IIIand CFT1 from chicken within an approximately 72 amino acid stretch. Asdenoted in FIG. 3A, this region is located in the proposed C-terminalcatalytic domains of FucTs. Therefore, we designated this gene asHpfucT.

[0111] The remaining sequence beyond this conserved region is relativelydivergent from that of eukaryotic FucTs. HpFucT appears to lack thetransmembrane segment that is common to eukaryotic FucTs, and which isusually located in their N-terminal region. On the other hand,eukaryotic FucTs do not contain the ˜100 aa region encompassing eight“DDLRV(or I)NY” repeats.

[0112] Characterization of the HpfucT gene product in E. coli cells Toinvestigate whether or not the predicted HpFucT gene represents acomplete locus, a maxicell system was used to detect the protein encodedby this gene. A modified CSR603 strain, which carries a Plac-controlledT7 DNA polymerase gene on the chromosome, was applied for the HpfucTexpression. Two recombinant plasmids, pBKHp763fucT38 (carrying thepartial HpFucT gene) and pBKHp763fucT39 (carrying the intact HpfucTgene), were constructed (FIG. 1). The HpfucT genes in these two plasmidswere controlled by a T7 promoter. Results of expression from theseplasmids are shown in FIG. 4. pBKHp763fucT39 gave rise to a specificproduct of ˜52 kDa (FIG. 4, lane 2) which is in agreement of thepredicted molecular mass of 54 kDa. In addition, a protein of ˜41 kDawas produced from pBKHp763fucT38-containing cells (FIG. 4, lane 1). Thesize of this product is consistent with the predicted 42 kDa of thetruncated HpFucT in which the C-terminal 101 amino acids of HpFucT wereremoved. In contrast, these HpfucT-encoded proteins were not produced incontrol cells containing either no plasmid or a vector without theinsert (FIG. 4, lanes 4 and 3, respectively). Two strong bands at ˜35kDa and 29 kDa were present in all the samples, indicating that theywere encoded by host genes. This evidence demonstrates that the clonedHpfucT represents a complete locus.

[0113] Biochemical assay of the overproduced HpFucT protein The partialsequence of this bacterial FucT is homologous to the catalytic domain ofmammalian FucTs, suggesting that HpFucT is a fucosyltransferase.Therefore, the nature of this enzyme activity was investigated. Todelineate the cellular location of the enzyme activity, membrane andcytoplasmic fractions of E. coli cells producing the HpFucT proteinswere prepared. The α1,3-HpFucT activity was quantitated using LacNAc-Ras an acceptor and GDP-fucose as the donor. Approximately 85% of thetotal enzyme activity was associated with the membrane fractioncontaining the intact HpFucT protein expressed from pBKHp763fucT39;whereas the remaining portion was present in the cytoplasmic fraction(Table 1). No detectable activity of either α1,2-FucT or α1,4-FucT wasfound in the samples tested. The Triton X-100-solubilized membranefraction gave rise to slightly higher α1,3-FucT activity than theuntreated extract (Table 1). No α1,3-FucT activity was obtained fromeither the membrane or the cytoplasmic fractions prepared from cellsproducing the truncated HpFucT protein encoded by pBKHp763fucT38. Thisresult indicated that the C-terminal 101 aa of HpFucT is crucial forfucosyltransferase activity.

[0114] The reaction products synthesized by the H. pylori α1,3fucosyltransferase were characterized using capillary electrophoresiswith laser-induced fluorescence detection of tetramethylrhodamine(TMR)-labeled acceptors as described in according to a known method. Thecapillary was 60 cm long (10 μm i.d.), and the samples were injectedonto the electrophoresis column at 1 kV for 5 seconds. The runningbuffer was 10 mM in phosphate, borate, phenylboronic acid, and SDS (pH9.3); the running voltage was 400 V/cm. The reaction mixture containingthe membrane fraction of cells harboring pBKHp763fucT39, GDP-fucose, andLacNAC-TMR produced a new peak (FIG. 5a, Lewis-X peak), whichco-migrated with a synthetic Le^(x)-TMR in the electropherogram (FIG.5c, peak 3), indicating that the new peak represents a Le^(x) productsynthesized by the bacterial α1,3-fucosyltransferase of this invention.Synthesis of Le^(x) with this enzyme was further tested by digestion ofLe^(x) with fucosidase, which cleaved the Le^(x) product and releasedLacNAc-TMR. Electrophoresis of the reaction mixture demonstrated thatthe concentration of LacNAc in the reaction mixture increased by 39%(FIG. 5b, LacNAc peak); whereas the concentration of the Le^(x) productdecreased by 36% (FIG. 5b, Le^(x) peak), showing that the test productwas synthesized by fucosyltransferase activity. FIG. 5C shows separationof nine standard TMR oligosaccharides found in mammalian metabolism,LacNAcb-(1), Fucα1→2Galβ1→4GlcNAcβ-(2), Galβ1→4[Fucα1→3]GlcNAcβ-(3),Fucα1→2Galβ1→4[Fucα1→3]GlcNAcβ-(4), GlcNAcβ-(5), linker arm-(6),NeuAcα2→6LacNAcβ-(7), NeuAcα2→3LacNAcβ-(8),NeuAcα2→3Galβ1→4[Fucα1→3]GlcNAcβ-TMR(9).

1 22 1 464 PRT Helicobacter pylori 1 Met Phe Gln Pro Leu Leu Asp Ala TyrVal Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro ProPro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu Ile LysGlu Phe Lys Asn Ser Val Leu Tyr 35 40 45 Phe Ile Leu Ser Gln Arg Tyr ThrIle Thr Leu His Gln Asn Pro Asn 50 55 60 Glu Phe Ser Asp Leu Val Phe GlyAsn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 Ile Leu Ser Tyr Gln Asn AlaLys Arg Val Phe Tyr Thr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe Asn LeuPhe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Asn Asp ArgTyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His Lys AlaGlu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130 135 140 Leu Lys Asp AsnSer Leu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe 145 150 155 160 Lys GluLys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp 165 170 175 ProLeu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185 190Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val 195 200205 Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn 210215 220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn225 230 235 240 Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp AlaTyr Phe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser ValAla Lys Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val His Asp PheLys Asn Phe Asp 275 280 285 Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His ThrHis Lys Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro Leu AsnThr Leu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu Ser PheLys Lys Ile Leu Ala Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn Asp ThrIle Tyr His Asp Asn Pro Phe Ile Phe 340 345 350 Cys Arg Asp Leu Asn GluPro Leu Val Thr Ile Asp Asp Leu Arg Val 355 360 365 Asn Tyr Asp Asp LeuArg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr 370 375 380 Asp Asp Leu ArgVal Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp 385 390 395 400 Leu ArgIle Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg 405 410 415 ValAsn Tyr Glu Arg Leu Leu Ser Lys Ala Thr Pro Leu Leu Glu Leu 420 425 430Ser Gln Asn Thr Thr Ser Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser 435 440445 Leu Pro Leu Leu Arg Ala Ile Arg Arg Trp Val Lys Lys Leu Gly Leu 450455 460 2 486 PRT Helicobacter pylori 2 Met Phe Gln Pro Leu Leu Asp AlaTyr Ile Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Ile Thr Ser Lys Ser ProPro Pro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu ValGlu Glu Phe Lys Lys Asn Ile Leu Tyr 35 40 45 Phe Ile Leu Ser Gln His TyrThr Ile Thr Leu His Gln Asn Pro Asn 50 55 60 Glu Pro Ser Asp Leu Val PheGly Ser Pro Ile Gly Ser Ala Arg Lys 65 70 75 80 Ile Leu Ser Tyr Gln AsnAla Lys Arg Val Phe Tyr Thr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe AsnLeu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Arg AspArg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His LysAla Glu Ser Val Asn Asp Thr Thr Ser Pro Tyr Lys 130 135 140 Leu Lys ProAsp Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His Phe 145 150 155 160 LysGlu Asn His Pro Asn Leu Cys Ala Val Val Asn Asn Glu Ser Asp 165 170 175Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185190 Pro Lys Arg Asn Ala Phe Tyr Asp Val Leu Asn Ser Ile Glu Pro Val 195200 205 Ile Gly Gly Gly Ser Val Lys Asn Thr Leu Gly Tyr Asn Ile Lys Asn210 215 220 Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe GluAsn 225 230 235 240 Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile AspAla Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro SerVal Ala Gln Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val Cys AspPhe Lys Asp Phe Asp 275 280 285 Glu Ala Ile Asp His Val Arg Tyr Leu HisThr His Pro Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro LeuAsn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu SerPhe Lys Lys Ile Leu Asp Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn AspThr Ile Tyr His Asp Asn Pro Phe Ile Phe 340 345 350 Tyr Arg Asp Leu AsnGlu Pro Leu Ile Ser Ile Asp Asp Asp Leu Arg 355 360 365 Val Asn Tyr AspAsp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn 370 375 380 Tyr Asp AspLeu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp 385 390 395 400 AspLeu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu 405 410 415Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val 420 425430 Asn Tyr Asp Asp Leu Arg Val Asn Tyr Glu Arg Leu Leu Gln Asn Ala 435440 445 Ser Pro Leu Leu Glu Leu Ser Gln Asn Thr Thr Phe Lys Ile Tyr Arg450 455 460 Lys Ala Tyr Gln Lys Ser Leu Pro Leu Leu Arg Ala Ala Arg LysLeu 465 470 475 480 Ile Lys Lys Leu Gly Leu 485 3 440 PRT Helicobacterpylori 3 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Lys1 5 10 15 Lys Met Pro Leu Ser Tyr Pro Pro Leu Lys Ile Ala Val Ala AsnTrp 20 25 30 Trp Gly Gly Ala Glu Glu Phe Lys Lys Ser Ala Met Tyr Phe IleLeu 35 40 45 Ser Gln Arg Tyr Thr Ile Thr Leu His Gln Asn Pro Asn Glu ProSer 50 55 60 Asp Leu Val Phe Gly Ser Pro Ile Gly Ala Ala Arg Lys Ile LeuSer 65 70 75 80 Tyr Gln Asn Thr Lys Arg Val Phe Tyr Ala Gly Glu Asn GluVal Pro 85 90 95 Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu LeuAsp Leu 100 105 110 Arg Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp ArgLeu His His 115 120 125 Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro TyrLys Ile Lys Pro 130 135 140 Asp Ser Leu Tyr Thr Leu Lys Lys Pro Ser HisHis Phe Lys Glu Lys 145 150 155 160 His Pro His Leu Cys Ala Val Val AsnAsp Glu Ser Asp Pro Leu Lys 165 170 175 Arg Gly Phe Ala Ser Phe Val AlaSer Asn Pro Asn Ala Pro Lys Arg 180 185 190 Asn Ala Phe Tyr Asp Ala LeuAsn Ser Ile Glu Pro Val Thr Gly Gly 195 200 205 Gly Ser Val Lys Asn ThrLeu Gly Tyr Lys Val Gly Asn Lys Asn Glu 210 215 220 Phe Leu Ser Gln TyrLys Phe Asn Leu Cys Phe Glu Asn Ser Gln Gly 225 230 235 240 Tyr Gly TyrVal Thr Glu Lys Ile Ile Asp Ala Tyr Phe Ser His Thr 245 250 255 Ile ProIle Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp Phe Asn Pro 260 265 270 LysSer Phe Val Asn Val His Asp Phe Lys Asn Phe Asp Glu Ala Ile 275 280 285Asp Tyr Val Arg Tyr Leu His Thr His Pro Asn Ala Tyr Leu Asp Met 290 295300 Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala Tyr Phe Tyr 305310 315 320 Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe Lys Thr IleLeu 325 330 335 Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Val Phe TyrArg Asp 340 345 350 Leu Asn Glu Pro Leu Val Ser Ile Asp Asp Leu Arg AlaAsp Tyr Asn 355 360 365 Asn Leu Arg Ala Asp Tyr Asn Asn Leu Arg Ala AspTyr Asn Asn Leu 370 375 380 Arg Ala Asp Tyr Asn Asn Leu Arg Ala Asp TyrAsp Arg Leu Leu Gln 385 390 395 400 Asn Arg Ser Pro Leu Leu Glu Leu SerGln Asn Thr Thr Phe Lys Ile 405 410 415 Tyr His Lys Ala Tyr His Lys SerLeu Pro Leu Leu Arg Ala Ile Arg 420 425 430 Arg Trp Val Lys Lys Leu GlyLeu 435 440 4 1670 DNA Helicobacter pylori CDS (145)...(1536) 4tctggcttgc acagctatgc cgcaggcgat cccttgccga tccctacttt cttatacttt 60ttggtagcga taccttttgc tcttgtgatc ttggcgtatt ttaaacgcca tttgagtttg 120cctaaattgg tttaaaggat aacc atg ttc caa ccc cta tta gac gct tat 171 MetPhe Gln Pro Leu Leu Asp Ala Tyr 1 5 gta gaa agc gct tcc att gaa aaa atggcc tct aaa tct ccc ccc ccc 219 Val Glu Ser Ala Ser Ile Glu Lys Met AlaSer Lys Ser Pro Pro Pro 10 15 20 25 cta aaa atc gct gtg gcg aat tgg tgggga gat gaa gaa att aaa gaa 267 Leu Lys Ile Ala Val Ala Asn Trp Trp GlyAsp Glu Glu Ile Lys Glu 30 35 40 ttt aaa aat agc gtt ctt tat ttt atc ctaagc caa cgc tac aca atc 315 Phe Lys Asn Ser Val Leu Tyr Phe Ile Leu SerGln Arg Tyr Thr Ile 45 50 55 acc ctc cac caa aac ccc aat gaa ttt tca gatctc gtc ttt ggt aac 363 Thr Leu His Gln Asn Pro Asn Glu Phe Ser Asp LeuVal Phe Gly Asn 60 65 70 ccc ctt gga tcg gcc aga aaa atc tta tcc tat caaaac gct aaa cga 411 Pro Leu Gly Ser Ala Arg Lys Ile Leu Ser Tyr Gln AsnAla Lys Arg 75 80 85 gtg ttt tac acc ggt gaa aac gaa tcg cct aat ttc aacctc ttt gat 459 Val Phe Tyr Thr Gly Glu Asn Glu Ser Pro Asn Phe Asn LeuPhe Asp 90 95 100 105 tac gcc ata ggc ttt gat gaa ttg gat ttt aat gatcgt tat ttg aga 507 Tyr Ala Ile Gly Phe Asp Glu Leu Asp Phe Asn Asp ArgTyr Leu Arg 110 115 120 atg cct tta tat tat gat agg cta cac cat aaa gccgag agc gtg aat 555 Met Pro Leu Tyr Tyr Asp Arg Leu His His Lys Ala GluSer Val Asn 125 130 135 gac acc act gcg ccc tac aaa ctc aaa gat aac agcctt tat gct tta 603 Asp Thr Thr Ala Pro Tyr Lys Leu Lys Asp Asn Ser LeuTyr Ala Leu 140 145 150 aaa aaa ccc tcc cat tgt ttt aaa gaa aaa cac cccaat tta tgc gca 651 Lys Lys Pro Ser His Cys Phe Lys Glu Lys His Pro AsnLeu Cys Ala 155 160 165 gta gtg aat gat gag agc gat cct ttg aaa aga gggttt gcg agc ttt 699 Val Val Asn Asp Glu Ser Asp Pro Leu Lys Arg Gly PheAla Ser Phe 170 175 180 185 gtc gcg agc aac cct aac gcc cct ata agg aacgct ttc tat gac gct 747 Val Ala Ser Asn Pro Asn Ala Pro Ile Arg Asn AlaPhe Tyr Asp Ala 190 195 200 cta aat tct att gaa cca gtt act ggg gga gggagc gtg aga aac act 795 Leu Asn Ser Ile Glu Pro Val Thr Gly Gly Gly SerVal Arg Asn Thr 205 210 215 tta ggc tat aac gtc aaa aac aaa aac gag ttttta agc caa tac aag 843 Leu Gly Tyr Asn Val Lys Asn Lys Asn Glu Phe LeuSer Gln Tyr Lys 220 225 230 ttc aac ctg tgt ttt gaa aac act caa ggc tatggc tat gta act gaa 891 Phe Asn Leu Cys Phe Glu Asn Thr Gln Gly Tyr GlyTyr Val Thr Glu 235 240 245 aaa atc att gac gct tac ttt agc cat acc attcct att tat tgg ggg 939 Lys Ile Ile Asp Ala Tyr Phe Ser His Thr Ile ProIle Tyr Trp Gly 250 255 260 265 agt cct agc gtg gcg aaa gat ttt aac cctaaa agt ttt gtg aat gtg 987 Ser Pro Ser Val Ala Lys Asp Phe Asn Pro LysSer Phe Val Asn Val 270 275 280 cat gat ttc aaa aac ttt gat gaa gcg attgac tat atc aaa tac ttg 1035 His Asp Phe Lys Asn Phe Asp Glu Ala Ile AspTyr Ile Lys Tyr Leu 285 290 295 cac acg cac aaa aac gct tat tta gac atgctt tat gaa aac cct ttg 1083 His Thr His Lys Asn Ala Tyr Leu Asp Met LeuTyr Glu Asn Pro Leu 300 305 310 aac acc ctt gat ggg aaa gct tac ttt taccaa aat ttg agt ttt aaa 1131 Asn Thr Leu Asp Gly Lys Ala Tyr Phe Tyr GlnAsn Leu Ser Phe Lys 315 320 325 aag atc cta gct ttt ttt aaa acg att ttagaa aac gat acg att tat 1179 Lys Ile Leu Ala Phe Phe Lys Thr Ile Leu GluAsn Asp Thr Ile Tyr 330 335 340 345 cac gat aac cct ttc att ttc tgt cgtgat ttg aat gag cct tta gta 1227 His Asp Asn Pro Phe Ile Phe Cys Arg AspLeu Asn Glu Pro Leu Val 350 355 360 act att gat gat ttg agg gtt aat tatgat gat ttg agg gtt aat tat 1275 Thr Ile Asp Asp Leu Arg Val Asn Tyr AspAsp Leu Arg Val Asn Tyr 365 370 375 gat gat ttg aga att aat tat gat gatttg agg gtt aat tat gat gat 1323 Asp Asp Leu Arg Ile Asn Tyr Asp Asp LeuArg Val Asn Tyr Asp Asp 380 385 390 ttg agg gtt aat tat gat gat ttg agaatt aat tat gat gat ttg agg 1371 Leu Arg Val Asn Tyr Asp Asp Leu Arg IleAsn Tyr Asp Asp Leu Arg 395 400 405 gtt aat tat gat gat ttg agg gtt aattat gag cgc ctc tta tca aaa 1419 Val Asn Tyr Asp Asp Leu Arg Val Asn TyrGlu Arg Leu Leu Ser Lys 410 415 420 425 gct acc cct ctt ttg gaa tta tcccaa aac acc act tct aaa atc tat 1467 Ala Thr Pro Leu Leu Glu Leu Ser GlnAsn Thr Thr Ser Lys Ile Tyr 430 435 440 cgc aaa gct tac caa aaa tcc ttacct ttg ttg cgc gcc ata agg aga 1515 Arg Lys Ala Tyr Gln Lys Ser Leu ProLeu Leu Arg Ala Ile Arg Arg 445 450 455 tgg gtt aaa aaa ttg ggt ttgtaaaattggg ggtaaactaa accccttgcg 1566 Trp Val Lys Lys Leu Gly Leu 460ctatcatcgc agacgctact tttctaaaac cagcgatatt agcccctaaa acaaaattat 1626gagggtcttt aaactcttta gcggtttgag agacattctt ataa 1670 5 476 PRTHelicobacter pylori 5 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu SerAla Ser Ile Glu 1 5 10 15 Lys Met Val Ser Lys Ser Pro Pro Pro Pro LeuLys Ile Ala Val Ala 20 25 30 Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu PheLys Lys Ser Val Leu 35 40 45 Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile ThrLeu His Gln Asn Pro 50 55 60 Asn Glu Ser Ser Asp Leu Val Phe Ser Asn ProLeu Gly Ala Ala Arg 65 70 75 80 Lys Ile Leu Ser Tyr Gln Asn Thr Lys ArgVal Phe Tyr Thr Gly Glu 85 90 95 Asn Glu Ser Pro Asn Phe Asn Leu Phe AspTyr Ala Ile Gly Phe Asp 100 105 110 Glu Leu Asp Phe Asn Asp Arg Tyr LeuArg Met Pro Leu Tyr Tyr Ala 115 120 125 His Leu His Tyr Glu Ala Glu LeuVal Asn Asp Thr Thr Ala Pro Tyr 130 135 140 Lys Leu Lys Asp Asn Ser LeuTyr Ala Leu Lys Lys Pro Ser His His 145 150 155 160 Phe Lys Glu Asn HisPro Asn Leu Cys Ala Val Val Asn Asp Glu Ser 165 170 175 Asp Leu Leu LysArg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn 180 185 190 Ala Pro MetArg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro 195 200 205 Val ThrGly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly 210 215 220 AsnLys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu 225 230 235240 Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr 245250 255 Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys260 265 270 Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn AsnPhe 275 280 285 Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His ProAsn Ala 290 295 300 Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr LeuAsp Gly Lys 305 310 315 320 Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys LysIle Leu Asp Phe Phe 325 330 335 Lys Thr Ile Leu Glu Asn Asp Thr Ile TyrHis Asn Asn Pro Phe Ile 340 345 350 Phe Tyr Arg Asp Leu His Glu Pro LeuIle Ser Ile Asp Asp Leu Arg 355 360 365 Val Asn Tyr Asp Asp Leu Arg ValAsn Tyr Asp Asp Leu Arg Val Asn 370 375 380 Tyr Asp Asp Leu Arg Val AsnTyr Asp Asp Leu Arg Val Asn Tyr Asp 385 390 395 400 Asp Leu Arg Val AsnTyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu 405 410 415 Arg Val Asn TyrAsp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val 420 425 430 Asn Tyr AspArg Leu Leu Gln Asn Ala Ser Pro Leu Leu Glu Leu Ser 435 440 445 Gln AsnThr Thr Phe Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser Leu 450 455 460 ProLeu Leu Arg Thr Ile Arg Arg Trp Val Lys Lys 465 470 475 6 425 PRTHelicobacter pylori 6 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu SerAla Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro Pro Pro LeuLys Ile Ala Val Ala 20 25 30 Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu PheLys Lys Ser Val Leu 35 40 45 Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile ThrLeu His Gln Asn Pro 50 55 60 Asn Glu Phe Ser Asp Leu Val Phe Ser Asn ProLeu Gly Ala Ala Arg 65 70 75 80 Lys Ile Leu Ser Tyr Gln Asn Thr Lys ArgVal Phe Tyr Thr Gly Glu 85 90 95 Asn Glu Ser Pro Asn Phe Asn Leu Phe AspTyr Ala Ile Gly Phe Asp 100 105 110 Glu Leu Asp Phe Asn Asp Arg Tyr LeuArg Met Pro Leu Tyr Tyr Ala 115 120 125 His Leu His Tyr Lys Ala Glu LeuVal Asn Asp Thr Thr Ala Pro Tyr 130 135 140 Lys Leu Lys Asp Asn Ser LeuTyr Ala Leu Lys Lys Pro Ser His His 145 150 155 160 Phe Lys Glu Asn HisPro Asn Leu Cys Ala Val Val Asn Asp Glu Ser 165 170 175 Asp Leu Leu LysArg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn 180 185 190 Ala Pro MetArg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro 195 200 205 Val ThrGly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly 210 215 220 AsnLys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu 225 230 235240 Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr 245250 255 Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys260 265 270 Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn AsnPhe 275 280 285 Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His ProAsn Ala 290 295 300 Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr LeuAsp Gly Lys 305 310 315 320 Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys LysIle Leu Asp Phe Phe 325 330 335 Lys Thr Ile Leu Glu Asn Asp Thr Ile TyrHis Lys Phe Ser Thr Ser 340 345 350 Phe Met Trp Glu Tyr Asp Leu His LysPro Leu Val Ser Ile Asp Asp 355 360 365 Leu Arg Val Asn Tyr Asp Asp LeuArg Val Asn Tyr Asp Arg Leu Leu 370 375 380 Gln Asn Ala Ser Pro Leu LeuGlu Leu Ser Gln Asn Thr Thr Phe Lys 385 390 395 400 Ile Tyr Arg Lys AlaTyr Gln Lys Ser Leu Pro Leu Leu Arg Ala Val 405 410 415 Arg Lys Leu ValLys Lys Leu Gly Leu 420 425 7 478 PRT Helicobacter pylori 7 Met Phe GlnPro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys MetAla Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp TrpGly Asp Glu Glu Ile Lys Glu Phe Lys Asn Ser Val Leu Tyr 35 40 45 Phe IleLeu Ser Gln Arg Tyr Thr Ile Thr Leu His Gln Asn Pro Asn 50 55 60 Glu PheSer Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 IleLeu Ser Tyr Gln Asn Ala Lys Arg Val Phe Tyr Thr Gly Glu Asn 85 90 95 GluSer Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120125 Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130135 140 Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe145 150 155 160 Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp GluSer Asp 165 170 175 Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser AsnPro Asn Ala 180 185 190 Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn SerIle Glu Pro Val 195 200 205 Thr Gly Gly Gly Ser Val Arg Asn Thr Leu GlyTyr Asn Val Lys Asn 210 215 220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys PheAsn Leu Cys Phe Glu Asn 225 230 235 240 Thr Gln Gly Tyr Gly Tyr Val ThrGlu Lys Ile Ile Asp Ala Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile TyrTrp Gly Ser Pro Ser Val Ala Lys Asp 260 265 270 Phe Asn Pro Lys Ser PheVal Asn Val His Asp Phe Lys Asn Phe Asp 275 280 285 Glu Ala Ile Asp TyrIle Lys Tyr Leu His Thr His Lys Asn Ala Tyr 290 295 300 Leu Asp Met LeuTyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr PheTyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe Lys 325 330 335 ThrIle Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe 340 345 350Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val 355 360365 Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr 370375 380 Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp385 390 395 400 Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp AspLeu Arg 405 410 415 Ile Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp LeuArg Val Asn 420 425 430 Tyr Glu Arg Leu Leu Ser Lys Ala Thr Pro Leu LeuGlu Leu Ser Gln 435 440 445 Asn Thr Thr Ser Lys Ile Tyr Arg Lys Ala TyrGln Lys Ser Leu Pro 450 455 460 Leu Leu Arg Ala Ile Arg Arg Trp Val LysLys Leu Gly Leu 465 470 475 8 454 PRT Helicobacter pylori 8 Met Phe GlnPro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys MetAla Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp TrpGly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Thr Leu Tyr 35 40 45 Phe IleLeu Ser Gln His Tyr Thr Ile Thr Leu His Arg Asn Pro Asp 50 55 60 Lys ProAla Asp Ile Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 IleLeu Ser Tyr Gln Asn Thr Lys Arg Ile Phe Tyr Thr Gly Glu Asn 85 90 95 GluSer Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110Leu Asp Phe Arg Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120125 Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130135 140 Ile Lys Gly Asn Ser Leu Tyr Thr Leu Lys Lys Pro Ser His Cys Phe145 150 155 160 Lys Glu Asn His Pro Asn Leu Cys Ala Leu Ile Asn Asn GluSer Asp 165 170 175 Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser AsnAla Asn Ala 180 185 190 Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn SerIle Glu Pro Val 195 200 205 Thr Gly Gly Gly Ala Val Lys Asn Thr Leu GlyTyr Lys Val Gly Asn 210 215 220 Lys Ser Glu Phe Leu Ser Gln Tyr Lys PheAsn Leu Cys Phe Glu Asn 225 230 235 240 Ser Gln Gly Tyr Gly Tyr Val ThrGlu Lys Ile Ile Asp Ala Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile TyrTrp Gly Ser Pro Ser Val Ala Lys Asp 260 265 270 Phe Asn Pro Lys Ser PheVal Asn Val His Asp Phe Asn Asn Phe Asp 275 280 285 Glu Ala Ile Asp TyrVal Arg Tyr Leu His Thr His Pro Asn Ala Tyr 290 295 300 Leu Asp Met LeuTyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr PheTyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe Lys 325 330 335 ThrIle Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile Phe 340 345 350Tyr Arg Asp Leu Asn Glu Pro Leu Val Ser Ile Asp Asn Leu Arg Ile 355 360365 Asn Tyr Asp Asn Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr 370375 380 Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp385 390 395 400 Leu Arg Ile Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Glu ArgLeu Leu 405 410 415 Gln Asn Ala Ser Pro Leu Leu Glu Leu Ser Gln Asn ThrSer Phe Lys 420 425 430 Ile Tyr Arg Lys Ile Tyr Gln Lys Ser Leu Pro LeuLeu Arg Val Ile 435 440 445 Arg Arg Trp Val Lys Lys 450 9 365 PRT Bostaurus 9 Met Tyr Pro Pro Gly Cys Ala Lys Val Lys Cys Ser Trp His His Cys1 5 10 15 Leu Pro Gly Leu Leu Leu Gln Leu Leu Leu Ala Leu Cys Phe PheSer 20 25 30 Tyr Leu Arg Met Ser Gln Glu Lys Pro Lys Pro Lys Pro Met TrpVal 35 40 45 Ser Glu Leu Gly Ala Pro Ser Gln Ala Thr Glu Gly Ser Ser AlaHis 50 55 60 Leu Pro Leu Arg Val Leu Leu Trp Thr Trp Pro Phe Asn Gln ProVal 65 70 75 80 Ala Leu Ser Arg Cys Ser Glu Leu Trp Pro Gly Thr Ala AspCys Gln 85 90 95 Leu Thr Val Asn Arg Ser Glu Tyr Pro Gln Ala Asp Ala ValPhe Val 100 105 110 His His Arg Glu Val Ser His Arg Pro Lys Met Gln LeuPro Pro Ser 115 120 125 Pro Arg Pro Ala Asp Gln Arg Trp Val Trp Phe SerMet Glu Ser Pro 130 135 140 Ser Asn Cys Leu Lys Leu Lys Asp Leu Asp GlyTyr Phe Asn Leu Thr 145 150 155 160 Met Ser Tyr Arg Arg Asp Ser Asp IlePhe Met Pro Tyr Gly Trp Leu 165 170 175 Glu Pro Trp Pro Ser Gln Pro ValGlu Thr Leu Leu Asn Ile Ser Ala 180 185 190 Lys Thr Lys Leu Val Ala TrpVal Val Ser Asn Trp Asn Thr Asp Ser 195 200 205 Ile Arg Val Gln Tyr TyrLys Leu Leu Lys Pro His Leu Gln Val Asp 210 215 220 Val Tyr Gly Arg PheHis Thr Pro Leu Pro His Ala Leu Met Ala Lys 225 230 235 240 Gln Leu SerGln Tyr Lys Phe Tyr Leu Ala Phe Glu Asn Ser Leu His 245 250 255 Pro AspTyr Ile Thr Glu Lys Leu Trp Lys Asn Ala Leu Gln Ala Trp 260 265 270 AlaVal Pro Val Val Leu Gly Pro Ser Arg Val Asn Tyr Glu Gln Phe 275 280 285Leu Pro Pro Lys Ala Phe Ile His Val Glu Asp Phe Gln Ser Pro Lys 290 295300 Asp Leu Ala Gln Tyr Leu Leu Ala Leu Asp Lys Asp Tyr Ala Ser Tyr 305310 315 320 Leu Asn Tyr Phe Arg Trp Arg Glu Thr Leu Arg Pro Arg Ser PheSer 325 330 335 Trp Ala Leu Met Phe Cys Lys Ala Cys Trp Lys Leu Gln GlnGlu Pro 340 345 350 Arg Tyr Gln Thr Val Pro Ser Ile Ala Ser Trp Phe Gln355 360 365 10 359 PRT Homo sapiens 10 Met Asp Pro Leu Gly Pro Ala LysPro Gln Trp Ser Trp Arg Cys Cys 1 5 10 15 Leu Thr Thr Leu Leu Phe GlnLeu Leu Met Ala Val Cys Phe Phe Ser 20 25 30 Tyr Leu Arg Val Ser Gln AspAsp Pro Thr Val Tyr Pro Asn Gly Ser 35 40 45 Arg Phe Pro Asp Ser Thr GlyThr Pro Ala His Ser Ile Pro Leu Ile 50 55 60 Leu Leu Trp Thr Trp Pro PheAsn Lys Pro Ile Ala Leu Pro Arg Cys 65 70 75 80 Ser Glu Met Val Pro GlyThr Ala Asp Cys Asn Ile Thr Ala Asp Arg 85 90 95 Lys Val Tyr Pro Gln AlaAsp Ala Val Ile Val His His Arg Glu Val 100 105 110 Met Tyr Asn Pro SerAla Gln Leu Pro Arg Ser Pro Arg Arg Gln Gly 115 120 125 Gln Arg Trp IleTrp Phe Ser Met Glu Ser Pro Ser His Cys Trp Gln 130 135 140 Leu Lys AlaMet Asp Gly Tyr Phe Asn Leu Thr Met Ser Tyr Arg Ser 145 150 155 160 AspSer Asp Ile Phe Thr Pro Tyr Gly Trp Leu Glu Pro Trp Ser Gly 165 170 175Gln Pro Ala His Pro Pro Leu Asn Leu Ser Ala Lys Thr Glu Leu Val 180 185190 Ala Trp Ala Val Ser Asn Trp Gly Pro Asn Ser Ala Arg Val Arg Tyr 195200 205 Tyr Gln Ser Leu Gln Ala His Leu Lys Val Asp Val Tyr Gly Arg Ser210 215 220 His Lys Pro Leu Pro Gln Gly Thr Met Met Glu Thr Leu Ser ArgTyr 225 230 235 240 Lys Phe Tyr Leu Ala Phe Glu Asn Ser Leu His Pro AspTyr Ile Thr 245 250 255 Glu Lys Leu Trp Arg Asn Ala Leu Glu Ala Trp AlaVal Pro Val Val 260 265 270 Leu Gly Pro Ser Arg Ser Asn Tyr Glu Arg PheLeu Pro Pro Asp Ala 275 280 285 Phe Ile His Val Asp Asp Phe Gln Ser ProLys Asp Leu Ala Arg Tyr 290 295 300 Leu Gln Glu Leu Asp Lys Asp His AlaArg Tyr Leu Ser Tyr Phe Arg 305 310 315 320 Trp Arg Glu Thr Leu Arg ProArg Ser Phe Ser Trp Ala Leu Ala Phe 325 330 335 Cys Lys Ala Cys Trp LysLeu Gln Glu Glu Ser Arg Tyr Gln Thr Arg 340 345 350 Gly Ile Ala Ala TrpPhe Thr 355 11 433 PRT Mus musculus 11 Met Ala Pro Ala Arg Gln Glu LeuGln His Glu Ser Arg Cys Arg Pro 1 5 10 15 Ser Arg Thr Val Asp Ala TrpArg Ala Ala Val Ala Thr Arg Gly Arg 20 25 30 His Met Glu Thr Pro Gly TyrArg Arg Arg Thr Arg Cys Gly Gly Trp 35 40 45 Gly Leu Pro Arg Ser Val SerSer Leu Ala Ala Val Gly Leu Leu Cys 50 55 60 Thr Ala Leu Thr Thr Phe IleCys Trp Gly Gln Leu Pro Pro Leu Pro 65 70 75 80 Trp Ala Ser Pro Ala ProGln Arg Leu Val Gly Val Leu Leu Trp Trp 85 90 95 Glu Pro Phe Arg Gly ArgGly Gly Tyr Pro Lys Ser Pro Pro Asp Cys 100 105 110 Ser Leu Arg Phe AsnIle Ser Gly Cys Arg Leu Leu Thr Asp Arg Ala 115 120 125 Ala Tyr Gly GluAla Gln Ala Val Leu Phe His His Arg Asp Leu Val 130 135 140 Lys Glu LeuHis Asp Trp Pro Pro Pro Trp Gly Ala Arg Glu Arg Thr 145 150 155 160 AspLys Ala Leu Val Leu Arg Val Phe Asp Asp Gln Glu Gly Ala Val 165 170 175Thr Leu Thr Gly Lys Ala Leu Glu Thr Val Gly Ser Arg Pro Pro Gly 180 185190 Gln Arg Trp Val Trp Met Asn Phe Glu Ser Pro Ser His Thr Pro Gly 195200 205 Leu Arg Gly Leu Ala Lys Asp Leu Phe Asn Trp Thr Leu Ser Tyr Arg210 215 220 Thr Asp Ser Asp Val Phe Val Pro Tyr Gly Phe Leu Tyr Ser ArgSer 225 230 235 240 Asp Pro Thr Glu Gln Pro Ser Gly Leu Gly Pro Gln LeuAla Arg Lys 245 250 255 Arg Gly Leu Val Ala Trp Val Val Ser Asn Trp AsnGlu His Gln Ala 260 265 270 Arg Val Arg Tyr Tyr His Gln Leu Ser Arg HisVal Ser Val Asp Val 275 280 285 Phe Gly Arg Thr Gly Pro Gly Arg Pro ValPro Ala Ile Gly Leu Leu 290 295 300 His Thr Val Ala Arg Tyr Lys Phe TyrLeu Ala Phe Glu Asn Ser Arg 305 310 315 320 His Val Asp Tyr Ile Thr GluLys Leu Trp Arg Asn Ala Phe Leu Ala 325 330 335 Gly Ala Val Pro Val ValLeu Gly Pro Asp Arg Ala Asn Tyr Glu Arg 340 345 350 Phe Val Pro Arg GlyAla Phe Ile His Val Asp Asp Phe Pro Asn Ala 355 360 365 Ala Ser Leu AlaAla Tyr Leu Leu Phe Leu Asp Arg Asn Val Ala Val 370 375 380 Tyr Arg ArgTyr Phe Arg Trp Arg Arg Ser Phe Ala Val His Ile Thr 385 390 395 400 SerPhe Trp Asp Glu Gln Trp Cys Arg Thr Cys Gln Ala Val Gln Thr 405 410 415Ser Gly Asp Gln Pro Lys Ser Ile His Asn Leu Ala Asp Trp Phe Gln 420 425430 Arg 12 356 PRT Gallus gallus 12 Met Glu Leu Gly Pro Arg Trp Ser ProAla Ala Arg Pro Gly Cys Pro 1 5 10 15 Arg Arg Trp Arg Arg Arg Trp AlaLeu Leu Gly Ala Leu Leu Gly Ala 20 25 30 Ala Leu Ala Leu Tyr Val Cys ValArg Glu Leu Arg Arg Arg Gly Ser 35 40 45 Ala Ala Gly Arg Pro Glu Gly GluVal Thr Val Leu Leu Trp Trp Glu 50 55 60 Pro Phe Gly Arg Pro Trp Arg ProAla Asp Cys Arg Arg Arg Tyr Asn 65 70 75 80 Ile Thr Gly Cys Leu Leu SerAla Asp Arg Gly Arg Tyr Gly Glu Ala 85 90 95 Arg Ala Val Leu Phe His HisArg Asp Leu Ala Leu His Gly Arg Gln 100 105 110 Gly Leu Pro Arg Gly ProPro Pro Arg Pro Pro Arg Gln Arg Trp Val 115 120 125 Trp Met Asn Phe GluSer Pro Ser His Ser Pro Gly Leu Arg Gly Leu 130 135 140 Ala Gly Leu PheAsn Trp Thr Met Ser Tyr Arg Arg Asp Ser Asp Val 145 150 155 160 Phe ValPro Tyr Gly Tyr Leu Tyr Glu Pro Pro Ser Pro Arg Pro Phe 165 170 175 ValLeu Pro Arg Lys Ser Arg Leu Val Ala Trp Val Ile Ser Asn Trp 180 185 190Asn Glu Glu His Ala Arg Val Arg Tyr Tyr Arg Gln Leu Lys Glu His 195 200205 Leu Pro Ile Asp Val Tyr Gly Ala Arg Gly Met Ala Leu Leu Glu Gly 210215 220 Ser Val Val Lys Thr Val Ser Ala Tyr Lys Phe Tyr Leu Ala Phe Glu225 230 235 240 Asn Ser Gln His Thr Asp Tyr Ile Thr Glu Lys Leu Trp LysAsn Ala 245 250 255 Phe Ala Ala Ser Ala Val Pro Val Val Leu Gly Pro ArgArg Ala Asn 260 265 270 Tyr Glu Arg Phe Ile Pro Ala Asp Ser Phe Ile HisVal Asp Asp Phe 275 280 285 Pro Ser Pro Arg Leu Leu Ala Thr Tyr Leu LysPhe Leu Asp Lys Asn 290 295 300 Lys Pro Ser Tyr Arg Arg Tyr Phe Ala TrpArg Asn Lys Tyr Glu Val 305 310 315 320 His Val Thr Ser Phe Trp Asp GluHis Tyr Cys Lys Val Cys Glu Ala 325 330 335 Val Arg Thr Ala Gly Asn GlnLeu Lys Thr Val Gln Asn Leu Ala Gly 340 345 350 Trp Phe Glu Ser 355 13372 PRT Helicobacter pylori 13 Met Phe Gln Pro Leu Leu Asp Ala Tyr ValGlu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro ProLeu Lys Ile Ala Val Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu Ile Lys GluPhe Lys Asn Ser Val Leu Tyr 35 40 45 Phe Ile Leu Ser Gln Arg Tyr Thr IleThr Leu His Gln Asn Pro Asn 50 55 60 Glu Phe Ser Asp Leu Val Phe Gly AsnPro Leu Gly Ser Ala Arg Lys 65 70 75 80 Ile Leu Ser Tyr Gln Asn Ala LysArg Val Phe Tyr Thr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe Asn Leu PheAsp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Asn Asp Arg TyrLeu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His Lys Ala GluSer Val Asn Asp Thr Thr Ala Pro Tyr Lys 130 135 140 Leu Lys Asp Asn SerLeu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe 145 150 155 160 Lys Glu LysHis Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp 165 170 175 Pro LeuLys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185 190 ProIle Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val 195 200 205Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn 210 215220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn 225230 235 240 Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala TyrPhe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val AlaLys Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe LysAsn Phe Asp 275 280 285 Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr HisLys Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn ThrLeu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu Ser Phe LysLys Ile Leu Ala Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn Asp Thr IleTyr His Asp Asn Pro Phe Ile Phe 340 345 350 Cys Arg Asp Leu Asn Glu ProLeu Val Thr Ile Asp Asp Leu Arg Val 355 360 365 Asn Tyr Asp Asp 370 1436 PRT Helicobacter pylori 14 Leu Arg Val Asn Tyr Asp Asp Leu Arg IleAsn Tyr Asp Asp Leu Arg 1 5 10 15 Val Asn Tyr Asp Asp Leu Arg Val AsnTyr Glu Arg Leu Leu Ser Lys 20 25 30 Arg Thr Pro Leu 35 15 36 PRTAabidoposis thaliana 15 Leu Glu Lys Asp Tyr Gly Val Leu Lys Thr Gln TyrAsp Ser Leu Arg 1 5 10 15 His Asn Phe Asp Ser Leu Arg Arg Asp Asn GluSer Leu Leu Gln Glu 20 25 30 Ile Ser Lys Leu 35 16 36 PRT Aabidoposisthaliana 16 Leu Glu Arg Asp Tyr Asp Leu Leu Lys Ser Thr Tyr Asp Gln LeuLeu 1 5 10 15 Ser Asn Tyr Asp Ser Ile Val Met Asp Asn Asp Lys Leu ArgSer Glu 20 25 30 Val Thr Ser Leu 35 17 36 PRT Aabidoposis thaliana 17Leu Glu Arg Asp Tyr Asp Leu Leu Lys Ser Thr Tyr Asp Gln Leu Leu 1 5 1015 Ser Asn Tyr Asp Ser Ile Val Met Asp Asn Asp Lys Leu Arg Ser Glu 20 2530 Val Thr Ser Leu 35 18 36 PRT Aabidoposis thaliana 18 Leu Glu Thr GluTyr Asn Ile Leu Arg Gln Asn Tyr Asp Asn Leu Ala 1 5 10 15 Ser Gln PheGlu Ser Leu Lys Lys Glu Lys Gln Ala Leu Val Ser Glu 20 25 30 Leu Gln ArgLeu 35 19 36 PRT Nicotiana tabacum 19 Leu Ala Ile Gln Val Gln Ser LeuThr Ala Glu Asn Asn Thr Leu Lys 1 5 10 15 Ser Glu Ile Asn Lys Leu MetGlu Asn Ser Glu Lys Leu Lys Leu Glu 20 25 30 Asn Ala Ala Leu 35 20 36PRT Petroselinum crispum 20 Leu Ala Ile Lys Val Asp Ser Leu Thr Ala GluAsn Met Ala Leu Lys 1 5 10 15 Ala Glu Ile Asn Arg Leu Thr Leu Thr AlaGlu Lys Leu Thr Asn Asp 20 25 30 Asn Ser Arg Leu 35 21 126 DNAHelicobacter pylori CDS (2)...(115) 21 t tat aag aat gtc tct caa acc gctaaa gag ttt aaa gac cct cat aat 49 Tyr Lys Asn Val Ser Gln Thr Ala LysGlu Phe Lys Asp Pro His Asn 1 5 10 15 ttt gtt tta ggg gct aat atc gctggt ttt aga aaa gta gcg tct gcg 97 Phe Val Leu Gly Ala Asn Ile Ala GlyPhe Arg Lys Val Ala Ser Ala 20 25 30 atg ata gcg caa ggg gtt tagtttacccc 126 Met Ile Ala Gln Gly Val 35 22 38 PRT Helicobacter pylori 22 TyrLys Asn Val Ser Gln Thr Ala Lys Glu Phe Lys Asp Pro His Asn 1 5 10 15Phe Val Leu Gly Ala Asn Ile Ala Gly Phe Arg Lys Val Ala Ser Ala 20 25 30Met Ile Ala Gln Gly Val 35

What is claimed is:
 1. A substantially purified transmembranesegment-free α1,3-fucosyltransferase polypeptide.
 2. The substantiallypurified transmembrane segment-free a 1,3-fucosyltransferase of claim 1,wherein the polypeptide catalyzes the synthesis ofGalβ1-4[Fucα1-3]GlcNAc (Lewis X) or NeuAcα2-3-Galβ1-4[Fucα1-3]GlcNAc(sialyl Lewis X).
 3. The polypeptide of claim 1, wherein the polypeptidelacks α1,4-fucosyltransferase activity.
 4. The polypeptide of claim 1,wherein the polypeptide lacks α1,2-fucosyltransferase activity.
 5. Thepolypeptide of claim 1, wherein the polypeptide lacksα1,4-fucosyltransferase and α1,2-fucosyltransferase activity.
 6. Thepolypeptide of claim 1, wherein the polypeptide has an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:
 3. 7. An isolated polynucleotide encoding thepolypeptide of claim
 1. 8. The polynucleotide of claim 7, wherein thesequence encodes the amino acid sequence selected from the group SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO:
 3. 9. A substantially purifiedtransmembrane segment-free α1,3-fucosyltransferase comprising apolypeptide having at least one repeat of the sequence comprisingX₁X₂LRX₃X₄Y, wherein X₁ is D or N; X₂ is D or N; X₃ is I, V or A; X₄ isN or D.